The polarized light microscope ("pol-scope") has the ability to measure submicroscopic molecular arrangements dynamically and non-destructively in living cells and other specimens. For this reason, it has been widely used in the field biological research, as set forth in the publications discussed below, which are incorporated herein by reference.
G. Valentin published the first observations on the appearance of parts of organisms between crossed nicols (Valentin, G. 1861. Die Untersuchung der Pflanzen- und der Thiergewebe in polarisiertem Lichte. Leipzig). The use of polarized light microscopy as applied to biology was marked by the numerous observations by W. J. Schmidt on the structure and development of skeletal and cellular components (Schmidt, W. J. 1924. Die Bausteine des Tierkorpers in polarisiertem tichte. Cohen, Bonn.; Schmidt, W. J. 1937. Die Doppelbrechung von Karyoplasma, Ztoplasma und Metaplasma. Borntrager, Berlin). Schmidt inferred the orientation of lipid molecules in membranes from observations in the polarized light microscope, before it was confirmed by X-ray diffraction. By carefully analyzing and eliminating sources of stray light in the polarizing microscope, Swann and Mitchison improved the sensitivity of the instrument considerably. (Swann M. M. and J. M. Mitchison. 1950. Refinements in polarized light microscopy. J. Exp. Biol. 27:226-237) and Inoue (Studies on depolarization of light at microscope lens surfaces. I. The origin of stray light by rotation at the lens surfaces. Exp. Cell Res. 3:199-208 ). With the improved sensitivity Inoue demonstrated the existence of fibers in the mitotic spindle directly in living cells (Inoue, Polarization optical studies of the mitotic spondle. I. The demonstration of spindle fibers in living cells. Chromosoma 5:487-500).
The introduction of the polarization rectifier increased the sensitivity further by a factor of 10 (Inoue, S. and W. L. Hyde. 1957. Studies on depolarization of light at microscope lens surfaces II. The simultaneous realization of high resolution and high sensitivity with the polarizing microscope. J. Biophys. Biochem. Cytol. 3:831-838), and led to a landmark study of DNA arrangement in living sperm (Inoue, S. and H. Sato. 1966. Deoxyribonucleic add arrangement in living sperm. In Molecular architecture in cell physiology. T. Nayashi and A. G. SzentGyorgyi, Editor. Prentice Hall, Englewood Cliffs, N.J. 209-248). However, it took Inoue and Sato three man-years to collect and analyze the data taken from three individual sperms with the high resolution, linear polarized light microscope.
Many of the pioneering studies in polarizing microscopy were associated with special instrumental developments to observe ever finer structural details and measure more rapidly specimen birefringence occurring in e.g. cytoplasmic flow or during rigor to relax transition in vertebrate striated muscle (Taylor, D. L. 1976. Quantitative studies on the polarization optical properties of striated muscle I. Birefringence changes of rabbit psoas muscle in the transition from rigor to relaxes state. J. Cell Biol. 68:497-511). Early automated detection schemes measured the magnitude of specimen birefringence in a single point (spot size 1.3 Micrometer diameter or larger, characteristic time constant 1/10 ms or longer) with an excellent sensitivity of a fraction of an Angstrom of specimen retardance. (Takasaki, H. 1961. Photoelectric measurement of polarized light by means of an ADP polarization modulator. I. Photoelectric Polarimeter, II. Photoelectric elliptic polarimeter. J. Opt. Soc. Am. 51:462-463) (Takasaki, H. 1961. Photoelectric measurement of polarized light by means of an ADP polarization modulator. III. Measurement of linear birefringence, IV. Lens interferometer. J. Opt. Soc. Am. 51:1146-1147); (Allen, R. D., J. Brault and R. D. Moore. 1963. A new method of polarization microscopic analysis I. Scanning with a birefringence detection system. J. Cell Biol. 18:223-235); (Taylor, D. L. and R M. Zeh. 1976. Methods for the measurement of polarization optical properties I. Birefringence. J. Microsc. 108:251-259); (Hiramoto, Y., Y. Hamaguchi, Y. Shoji and S. Shimod. 1981. Quantitative studies on the polarization optical properties of living cells I. Microphotometric birefringence detection system. J. Cell Biol. 89:115-120) However, the low spatial resolution, especially the restriction to a single point or area, and the fixed orientation of the measured birefringence limited the use of these detectors.
The restriction of the measurement of optical anisotropy to a single specimen point was overcome by Tinoco and collaborators in a structural study of spermatocyte nuclei, by introducing a scanning stage in their differential polarization microscope (Mickols, W., M. F. Maestre and I. Tinoco Jr. 1987. Differential polarization microscopy of changes in structure in spermatocyte nuclei. Nature 328:452-454; Oldenbourg, R. 1991. Analysis of edge birefringence. Biophys. J. accepted for publication). The optical path in this instrument is similar to a transmission, stage scanning confocal microscope. While the spatial resolution and sensitivity is high, the time required to obtain a complete image is about 45 minutes.
The use of video cameras to record images of birefringent specimens with the polarized light microscope was first introduced by Allen and collaborators (Allen, R. D., J. L. Travis, N. S. Allen and Ho Yilmaz. 1981. Video-enhanced contrast polarization (AVEC-POL) microscopy: A new method applied to the detection of birefringence in the motile reticulopodial network Allogromia laticollaris. Cell Motil. 1:275-289) and by Inoue (1981. Video image processing greatly enhances contrast, quality and speed in polarization-based microscopy. J. Cell Biol. 89:346-356). To measure specimen birefringences from recorded images one can use a predetermined calibration curve to relate measured intensities to specimen birefringences in different parts of the image (Schaap, C. J. and A. Forer. 1984. Video digitizer analysis of birefringence along the length of single chromosomal spindle fibres I. Description of the system and general results.]. Cell Sci. 65:21-40). This method is relatively fast, but subject to errors from variations in light intensities that stem from other sources than birefringence, e.g. light scattering or shading. In a study on edge birefringence, Oldenbourg recorded images of a given specimen at several different compensator settings and used the stack of images to compute the specimen birefringences independent of the overall intensity and the background light in different parts of the viewing field. Oldenbourg, R. 1991. Analysis of edge birefringence. Biophys. J. Vol. 60 page 629.
With the traditional pol-scope, thus, single images display only those anisotropic structures that have a limited range of orientations with respect to the polarization axes of the microscope. Furthermore, rapid measurements are restricted to a single image point or single area that exhibits uniform birefringence or other form of optical anisotropy, while measurements comparing several image points take an inordinately long time.
There remains a need for a pol-scope that will permit data collection and determination of anisotropic structures (e.g., specimen birefringence) irrespective of orientation and over a wide range of magnitude, and that will do so in a short period of time.